PURINE CATABOLISM IN DROSOPHZLA MELANOGASTER. 11. GUANINE DEAMINASE, INOSINE PHOSPHORYLASE AND ADENOSINE DEAMINASE ACTIVITIES IN MUTANTS WITH ALTERED XANTHINE DEHYDROGENASE ACTIVITIES1 L. D. HODGEZ AND E. GLASSMANS Department of Biochemistry and the Genetics Curriculum, University of North Carolina at Chapel Hill, North Carolina 27514 Received May 23, 1967 variety of mutants affect purine metabolism in Drosophila mlanogaster. A GEER(1963,1964) has reported a strain with a polygenic nutritional require- ment for yeast RNA. DANNEELand ESCHRICH-ZIMMERMANN(1957) report that bw (brown) and w (white) mutants excrete excess uric acid. The most exten- sively studied are those mutants that control xanthine dehydrogenase (XDH) (GLASSMAN1965) : lxd (low xanthine dehydrogenase) at locus 331- on the third chromosome (KELLERand GLASSMAN19641, ry (rosy eye color) at locus 52* on the third chromosome (BRIDGESand BREHME1944) and ma4 (maroon-like eye color) at locus 64+ on the X chromosome (HUBBYand FORREST1960). Various mutant alleles of ry and ma-1 have no detectable XDH activity (FORREST,GLASS- MAN and MITCHELL1956), while Zxd has approximately 20 to 25% normal XDH activity (KELLERand GLASSMAN1964). The ry locus is a structural gene for XDH (KARAMand GLASSMAN1967; YEN and GLASSMAN1965). It would be of interest to determine if mutants affecting XDH have any effect on the remainder of the pathway of purine catabolism. This could come about in various ways. For example, the genes coding for these enzymes might be coordinately controlled by a regulatory gene. Since the enzymes of this pathway react with purines, and if ma-1+, ry+, or lxd+ control a polypeptide common to other enzymes of purine catabolism, coordinate control might be present. Three enzymes close to XDH in the pathway of purine catabolism were chosen for this study; adenosine deami- nase, inosine phosphorylase, and guanine deaminase (HODGEand GLASSMAN 1967). Some of their properties have been elucidated and the levels of their activities in these mutants have been ascertained. MATERIALS AND METHODS Chemicals: Crystalline bovine plasma albumin, the protein standard solution (IO mg protein nitrogen per ml) was purchased from the Armour Pharmaceutical Company. Guanine and milk xanthine oxidase were from the California Corporation for Biochemical Research. Norite A and ' Part of the work reported here was supported by Public Health Service Grant GM-08202. ' Supported by Genetics Research Training Grant, 5TI-GH-685, from the Public Health Service. Present Address: Department of Cell Biologg-, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461. Supported by a Research Career Development Award, GbI-K3-14,911, from the Public Health Senrice. Genetics 57: 571-577 Noveniher 19b7. 5 72 L. D. HODGEAND E. GLASSMAN Folin-Ciocalteu Reagent were from the Fisher Scientific Company. Adenosine, deoxyadenosine, inosine and deoxyinosine were from Schwarz BioResearch, Inc. Tris( hydroxymethyl) amino- methane was from the Sigma Chemical Company. The following abbreviations appear in the text: Tris = Tris( hydro'xymethyl) aminomethane-HCL; Tris-albumin = Tris containing 1 mg per ml of crystalline bovine plasma albumin. D. melanogaster strains: The following were used (BRIDGESand BREHME1944): Pac, Pacific wild-type; CS, Canton-S wild type containing scarlet eye (3-444); w ec containing white eye (1-15+) and echinus (1-5 5+); ru lxd by, containing roughoid eye (3-O+), low xanthine dehydrogenase (3-33k) and blistery wing (3-48t); st ry containing scarlet eye (3-444)and rosy eye (3-52 4) ; U f B9 mal-l containing vermilion eye ( 1-33zk ), forked bristles ( 1-56+ ) , Beadex wing (1-594) and maroon-like eye (1-64t); bw, brown eye (2-104.k). The Canton-S wild-type into which st had been introduced was a gift o'f DR. B. W. GEER.All larvae were raised at 25 i- 1"C on a standard media ( GLASSMANand MCLEAN1962). Preparation of Extracts: All extracts were prepared at temperatures below 5°C. Third-instar larvae were homogenized in a Kontes Dual1 glass tissue grinder for approximately one minute using 1.5 g of larvae per ml of buffer solution. Homogenates were centrifuged at 34,000 g for 20 minutes in a Sorvall -RC-2 centrifuge. The supernatant solution was removed from beneath the overlying fatty layer and filtered through glasswool. Using the methods described elsewhere (HODGEand GLASSMAN1967), this extract was either treated with charcoal (CHAR-crude) or used to make a pH5 supernatant solution which then was treated with charcoal (CHAR-pH5). Enzyme assays: Assays are modifications of previously published procedures (PRICE,OTEY and PLESNER1956; KAPLAN1956; SHUSTER1956) and were performed in triplicate in a Beckman DB Spectrophotometer. The change in optical density of a reaction mixture lacking substrate was used as a control. Assays were performed at 30°C using buffers and substrate solution kept at this temperature. Each enzyme was assayed at the optimal pH, at a concentration of the substrate which saturated the enzyme, in the range where reaction rate is proportional to enzyme concentration, and with amounts of enzyme that gave linear reaction rates for the entire time of the assay. The assays are based upon measurements of the initial rate of reaction such that the kinetics approached zero order. Enzymatic units and specific activities are defined according to the recommendations of the Commission on Enzymes (1961 ) . To assay guanine deaminase, larvae were homogenized in cold 0.1 M Tris, pH 7.0 and CHAR-pH5 was prepared. -2.8 ml of 0.1 M Tris-albumin, pH 7.3; 0.15 ml of 3 x le3M guanine, and 0.1 ml of freshly prepared CHAR-pH5 were mixed rapidly. The decrease in optical density at 245 mp was followed for 10 minutes. The optimum pH was 7.3 and a 50% reduction in activity occurred at pH 6.6 and at 8.4. The reaction rate was proportional to the amwnt of protein added between 0.075 to 0.60 mg. This preparation did not deaminate guanosine, deoxy- guanosine, GMP or GTP. A unit of guanine deaminase is the amount of enzyme that catalyzes one micromole of guanine per minute. To assay inosine phosphorylase, larvae were homogenized in 0.1 M sodium phosphate buffer, pH 7.5, and CHAR-crude was prepared. 1.8 ml of 0.05 M phosphate buffer, pH 6.8, 1.0 ml of 10-3 M inxine, 0.1 ml of milk xanthine oxidase (0.02 units), and 0.1 ml of extract were mixed rapidly. The increase in optical density at 290 mp was followed for 90 minutes using inosine as substrate and 60 minutes using deoxyinosine as substrate. Deoxyinosine was split at a slightly higher rate than inosine in all strains, especially by extracts of the Pac strain. The reaction rate was propxtional to the amount of protein added between 0.2 and 0.8 mg. 0.1 M or 0.005 M sodium phosphate buffer gave a maximum of activity suggesting that this enzyme is a phos- phorylase. No activity was obtained with Tris buffer. The use of milk xanthine oxidase avoided difficulties when assaying activity in the XDH deficient mutants, ma-1, ry and lxd. A unit of inosine phosphorylase is the amount of enzyme that catalyses one micromole of inosine per minute. To assay adenosine deaminase larvae were homogenized in cold 0.05 M succinate buffer, pH 6.8, and CHAR-pH5 prepared. 2.8 ml of 0.05 M succinate buffer, pH 6.8, 0.3 ml of 1.5 x le3 M adenosine or deoxyadenosine solution, and 0.8 ml of extract were rapidly mixed. The decrease in optical density at 265 mp was recorded for 20 minutes. The activity exhibited a broad. flat DROSOPHILA PURINE CATABOLISM 5 73 naximum at pH 6.9 with a 15% reduction in activity at pH 6.1 and 34% reduction at pH 8.6. The reaction rate was proportional to added protein between 0.1 to 0.6 mg. Adenine, AMP and ATP were not deaminated. Deoxyadenosine was deaminated at approximately half the rate as adenosine by all strains. Trace activity noted with AMP was probably due to adenosine derived from a breakdown of the AMP. A unit of adenosine deaminase is the amount of enzyme that catalyzes one micromole of adenosine per minute. Protein determinatioe Protein was determined by a modification of the technique of LOWRY et al. (1951). RESULTS Table 1 shows the averages of replicate determination of the activities of guanine deaminase in seven strains. All extracts possessed guanine deaminase activity. Three strains, ma-2, bw and wee, possess lower amounts of guanine deaminase activity, particularly the extracts of the bw strain which has only 50% of the activity of the Pacific wild type (Pac) . The txd and ry genes appear to have no effect on the levels of activity of this enzyme. The Canton-S (CS) strain which previously appeared by autoradiographic assay methods to be unable to convert guanine to xanthine (HODGEand GLASSMAN1967) had guanine deaminase activity equal to Pac. In an attempt to understand the differences between previous results (HODGE and GLASSMAN1967) which indicated that extracts of the CS strain had little guanine deaminase and the present results, extracts of Pac and CS were incubated for various times at 30°C with occasional shaking. Under these conditions the CS extract showed a progressive loss of guanine deamniase activity of approximately 80% by 60 minutes, but the Pac extracts lost only about 25% in the same time (Figure 1, left). Mixtures of Pac and CS extracts are inactivated to the same degree as CS extracts suggesting that an inactivator of guanine deaminase activity is present in the CS extracts. Figure 1, right, shows that boiling inactivates this factor. It was noted that extracts of the CS strain became black during the incu- bation, suggesting that tyrosinase activity is very high.